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Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Alasmari W, Costello S, Correia J, Oxenham SK, Morris J, Fernandes L, Ramalho-Santos J, Kirkman-Brown J, Michelangeli F, Publicover S, Barratt CL - J. Biol. Chem. (2013)

Bottom Line: Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose.The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not.We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive and Developmental Biology, Medical School, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

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Stored Ca2+ but not activation of CatSper induces hyperactivation.A and B, increment in percentage of hyperactivated cells induced by 25 mm NH4Cl (dark blue), 20 and 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). A, cells prepared by swim-up into sEBSS; B, cells prepared by density gradient centrifugation into STF. Each bar shows mean ± S.E. (error bars) of 20–60 experiments except for thimerosal swim-up (n = 8), TMA (n = 4–5). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control. C and D, frequency distributions of ALH (C) and linearity (D) under control conditions (black) and after stimulation with 25 mm NH4Cl (dark blue), 2 mm 4-AP (green), and 5 μm thimerosal (red). Each plot shows mean ± S.E. of 19 (control, 4-AP, progesterone), 9 (NH4Cl), and 10 (thimerosal) STF samples. E–H, example tracks of control (E) and cells exposed to 20 mm TMA (F), 2 mm 4-AP (G), and 5 μm thimerosal (H). All traces start at the origin (0, 0), and scales show distance (μm). Sample rate = 100 Hz.
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Figure 2: Stored Ca2+ but not activation of CatSper induces hyperactivation.A and B, increment in percentage of hyperactivated cells induced by 25 mm NH4Cl (dark blue), 20 and 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). A, cells prepared by swim-up into sEBSS; B, cells prepared by density gradient centrifugation into STF. Each bar shows mean ± S.E. (error bars) of 20–60 experiments except for thimerosal swim-up (n = 8), TMA (n = 4–5). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control. C and D, frequency distributions of ALH (C) and linearity (D) under control conditions (black) and after stimulation with 25 mm NH4Cl (dark blue), 2 mm 4-AP (green), and 5 μm thimerosal (red). Each plot shows mean ± S.E. of 19 (control, 4-AP, progesterone), 9 (NH4Cl), and 10 (thimerosal) STF samples. E–H, example tracks of control (E) and cells exposed to 20 mm TMA (F), 2 mm 4-AP (G), and 5 μm thimerosal (H). All traces start at the origin (0, 0), and scales show distance (μm). Sample rate = 100 Hz.

Mentions: For obtaining detailed tracks (e.g.Fig. 2, E–H), cells were prepared as described in method 1, diluted to ∼0.5 × 106 cells/ml, and viewed in a 20-μm depth chamber using a Hamamatsu Photonics C9300 CCD camera at 100 Hz. The stage was maintained at 37 °C (LINKAM C0102 stage heater).


Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Alasmari W, Costello S, Correia J, Oxenham SK, Morris J, Fernandes L, Ramalho-Santos J, Kirkman-Brown J, Michelangeli F, Publicover S, Barratt CL - J. Biol. Chem. (2013)

Stored Ca2+ but not activation of CatSper induces hyperactivation.A and B, increment in percentage of hyperactivated cells induced by 25 mm NH4Cl (dark blue), 20 and 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). A, cells prepared by swim-up into sEBSS; B, cells prepared by density gradient centrifugation into STF. Each bar shows mean ± S.E. (error bars) of 20–60 experiments except for thimerosal swim-up (n = 8), TMA (n = 4–5). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control. C and D, frequency distributions of ALH (C) and linearity (D) under control conditions (black) and after stimulation with 25 mm NH4Cl (dark blue), 2 mm 4-AP (green), and 5 μm thimerosal (red). Each plot shows mean ± S.E. of 19 (control, 4-AP, progesterone), 9 (NH4Cl), and 10 (thimerosal) STF samples. E–H, example tracks of control (E) and cells exposed to 20 mm TMA (F), 2 mm 4-AP (G), and 5 μm thimerosal (H). All traces start at the origin (0, 0), and scales show distance (μm). Sample rate = 100 Hz.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585060&req=5

Figure 2: Stored Ca2+ but not activation of CatSper induces hyperactivation.A and B, increment in percentage of hyperactivated cells induced by 25 mm NH4Cl (dark blue), 20 and 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). A, cells prepared by swim-up into sEBSS; B, cells prepared by density gradient centrifugation into STF. Each bar shows mean ± S.E. (error bars) of 20–60 experiments except for thimerosal swim-up (n = 8), TMA (n = 4–5). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control. C and D, frequency distributions of ALH (C) and linearity (D) under control conditions (black) and after stimulation with 25 mm NH4Cl (dark blue), 2 mm 4-AP (green), and 5 μm thimerosal (red). Each plot shows mean ± S.E. of 19 (control, 4-AP, progesterone), 9 (NH4Cl), and 10 (thimerosal) STF samples. E–H, example tracks of control (E) and cells exposed to 20 mm TMA (F), 2 mm 4-AP (G), and 5 μm thimerosal (H). All traces start at the origin (0, 0), and scales show distance (μm). Sample rate = 100 Hz.
Mentions: For obtaining detailed tracks (e.g.Fig. 2, E–H), cells were prepared as described in method 1, diluted to ∼0.5 × 106 cells/ml, and viewed in a 20-μm depth chamber using a Hamamatsu Photonics C9300 CCD camera at 100 Hz. The stage was maintained at 37 °C (LINKAM C0102 stage heater).

Bottom Line: Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose.The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not.We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive and Developmental Biology, Medical School, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

Show MeSH
Related in: MedlinePlus